Human immunodeficiency virus (HIV), previously named human T-cell lymphotropic virus-III (HTLV-III), a human retrovirus has been found to be the causative agent of the acquired immunodeficiency syndrome (AIDS).sup.1.
AIDS is a devastating, death threatening disease and has spread alarmingly in Africa, the Carribean basin, Unites States, Europe and throughout the world. Based on existing knowledge, the disease is spread through sexual contact, transfusion of contaminated blood, and sharing of unsterilized needles. The mortality rate of the disease is very high, approximately 70% of those who manifest symptoms of the disease have died, no effective or preventive measures against the infection by HIV has been found. It is, therefore, imperative that the population be educated on the dangers of the disease, and the manner of transmission. Most importantly, the blood supply for those who are in need of blood transfusions, such as hemophiliacs and surgical patients, must be screened to eliminate possible contamination by HIV.
Heretofore, the presence of HIV has been determined indirectly. That is, screening tests for AIDS, AIDS Related Complex (ARC) and pre-AIDS conditions have been based on the use of the deactivated virus.sup.2 or a synthetic peptide composition.sup.3 described in co-pending applications Ser. Nos. 847,102 and 13,014 to detect the presence of antibodies to HIV. From the presence of antibodies to HIV, it is inferred that the sample biological body fluid would contain the antigen, HIV.
Detection of HIV itself has been principally based on the rather cumbersome reverse transcriptase assay.
In 1985, J. S. McDougal et al. reported a sandwich enzyme-linked immunoassay (ELISA) for detecting HIV.sup.4. The method involved the collection of IgG fraction from a serum sample with high titer antibody to HIV obtained from a homosexual with chronic unexplained lymphadenopathy. The IgG fraction was purified by ammonium sulfate precipitation and diethylaminoethyl cellulose chromotography. A portion of the IgG fraction was coupled with fluorescien isothiocyanate (FITC). Another portion was coupled to horseradish peroxidase (HRPO).
The FITC-anti-HIV at 19 ug/ml was incubated with HIV in disrupted cultured cells and found to react specifically with HIV and become stained. The entire process takes at least about two to three hours.
Alternatively, an IgG fraction was used to coat a 96-well microtiter tray. HIV cultured cell supernates were mixed with buffer and added to the wells and incubated at 4.degree. C. overnight. HRPO-anti-HIV was added to each well and caused to react with o-phenylene-diamine (OPD) to give a colored product. The entire ELISA procedure takes approximately one day.
Another method for direct detection of HIV has been reported.sup.5. This method utilizes rabbit polyclonal anti-HIV fixed on microtiter plate wells to capture the HIV p24 protein. The captured protein is complexed with biotinylated rabbit polyclonal anti-HIV p24 and probed with a streptavidin-horseradish peroxidase conjugate. The complex is then detected by incubation with orthophenyl-diamine to produce a yellow color in positive wells.
The method calls for immunization of rabbits with HIV, harvesting and purifying the rabbit anti-HIV polyclonal antibodies which is very time consuming.
Agglutination tests using latex beads are known. For example, agglutination has been used in diagnostic tests to determine pregnancy.sup.6 and measles.sup.7.
However, up to the present, agglutination has not been found to be applicable as a means for the diagnosis of HIV infection in body fluids.
Up to the present, no rapid simple assays have been developed for the direct detection of HIV.
It is the object of the present invention to develop a rapid and simple screening test for the direct determination of HIV in body fluids.